The anaerobic spore-forming bacilli of the genus Clostridium represent a major class of human pathogens, causing, inter alia, botulism, tetanus and gas gangrene. For the most part, the pathogenicity depends largely on the release of extremely toxic exotoxins or highly destructive enzymes.
One of the most lethal and necrotizing toxins produced by many of the Clostridium species associated with invasive infection in humans is a calcium-dependent enzyme lecithinase, known as Phospholipase C (EC3.1.4.3). This enzyme catalyzes the breakdown of lecithin (choline phosphoglyceride) in cell membranes to diglyceride and phosphorylcholine, as well as the hydrolysis of cephalin and sphingolmyelin. Primarily through its action on lecithin, which is present in membranes of many cells, the toxin can cause extensive damage in a variety of animal tissues. (See, generally, Mollby, R., "Bacterial Phospholipases," in Bacterial Toxins and Cell Membranes, Eds. Jeljaszewicz and Wadstrom (1977), which is incorporated herein by reference.)
Although much research has been conducted on the various activities of purified Clostridium Phospholipase C (see, e.g., Yamakama and Ohsaka, J. Biochem 81:115-126 (1977), which is incorporated herein by reference, little is known about its structure (e.g., amino acid sequence). This is in part due to the risks associated with the production of large quantities of substantially pure Phospholipase C, which can necessitate growing large volumes of typically toxic Clostridium bacteria. Commonly used processes for purification do not, of course, guarantee removal of other Clostridium toxins. Also, most assays for Phospholipase C are time consuming and difficult to perform with high degrees of accuracy and reproducibility.
In general, the species diagnosis of most Clostridium infections is complicated by the fact that many such infections contain more than one type of bacteria. This diagnosis could rely on the detection of particular species variants of Phospholipase C. In this regard, isolating genes encoding Clostridium Phospholipase C is desirable, particularly in view of the cloning and expression of Phospholipase genes from other virulent bacteria species (e.g., P. aeruginosa, Pritchard and Coleman, J. Bacterial. 167:291-298 (1986) and S. aureus, Coleman et al., Microb. Path. 1:549-564 (1986), both of which are incorporated herein by reference.)
Thus, there exists a need for the safe and economic production of substantial quantities of Clostridium Phospholipase C, as well as new and improved assays for the toxin. Ideally, the material will be substantially free of other, naturally-occurring Clostridium proteins The present invention fulfills these needs.